Method of promoting production of living tissue equivalents

ABSTRACT

The present invention provides methods, improved cell culture medium and kits for accelerating the generation of tissue equivalents, and for improving the quality of tissue equivalents, by growth in the presence of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments and analogues thereof and/or AII AT 2  type 2 receptor agonists.

CROSS REFERENCE

[0001] This application is a continuation in part of U.S. Application Serial No. 60/077,499 filed Mar. 11, 1998 and 60/089,064 filed Jun. 12, 1998.

FIELD OF THE INVENTION

[0002] This present invention relates to methods, tissue culture medium, and kits for accelerating the production living tissue equivalents.

BACKGROUND OF THE INVENTION

[0003] Recently, various systems for the in vitro production of tissue equivalents have been described. As used herein, the term “tissue” comprises any group or layer of cells which together perform one or more certain functions. Such tissue equivalents include, but are not limited to, equivalents of epithelial tissue, connective tissue, cartilage, bone, organs, vascular grafts, glands and blood vessels and comprise living cells and extracellular matrix molecules, principally collagen, and may optionally be provided with components not typically found in normal tissue.

[0004] Three-dimensional cell culture systems have been described which can be used to culture a variety of different cells and tissues in vitro for prolonged periods of time (U.S. Pat. Nos. 5,624,840; 5,541,107; 5,521,087; 5,516,681, 5,516,680; 5,512,475, herein incorporated by reference in their entirety). Cells derived from a desired tissue are inoculated and grown on a pre-established stromal support matrix. The stromal support matrix comprises stromal cells, such as fibroblasts, actively growing on a three-dimensional matrix. Stromal cells may also include other cells found in loose connective tissue such as endothelial cells, macrophages/monocytes, adipocytes, pericytes, reticular cells found in bone marrow stroma, etc. The stromal matrix provides the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts found in vivo.

[0005] These inventions are based, in part, on the discovery that growth of stromal cells in three dimensions will sustain active proliferation of cells in culture for longer periods of time than will monolayer systems (U.S. Pat. No. 5,510,254). This may be due, in part, to the increased surface area of the three-dimensional matrix which results in a prolonged period of active proliferation of stromal cells. These proliferating stromal cells elaborate proteins, growth factors and regulatory factors necessary to support the long term proliferation of both stromal and tissue-specific cells inoculated onto the stromal matrix. In addition, the three-dimensionality of the matrix allows for a spatial distribution which more closely approximates conditions in vivo, thus allowing for the formation of microenvironments conducive to cellular maturation and migration. The growth of cells in the presence of this support may be further enhanced by adding proteins, glycoproteins, glycosaminoglycans, a cellular matrix, and other materials to the support itself or by coating the support with these materials.

[0006] Similarly, tissue equivalents comprising a hydrated collagen lattice contracted by a contractile agent, such as fibroblast cells or blood platelets, in combination with a variety of cell types to form the tissue equivalent are disclosed in U.S. Pat. Nos. 4,485,096; 4,485,097; 4,539,716; 4,546,500; 4,604,346; and 4,835,102; 4,837,379; 5,256,418; 5,536,656; and RE 35,399, all of which are incorporated herein by reference.

[0007] In specific embodiments of the invention, bone marrow, bone, skin, dermis, liver, kidney, cartilage, ligament, tendon, pancreas, and heart valve tissues may be grown in the three dimensional culture system. The resulting cultures have a variety of applications ranging from transplantation or implantation, in vivo, of cells grown in the cultures, cytotoxicity testing and screening compounds in vitro, and the design of “bioreactors” for the production of biological materials in vitro. For example, skin tissue equivalents can be used as grafts to treat bum victims or ulcer patients, while kidney and liver tissue equivalents can be used for transplanting where disease has caused organ damage or failure. For diffuse tissues such as bone marrow, the proliferating cells could be isolated from the culture system for transplantation. Tendon, ligament, and cartilage tissue equivalents can be used for transplantation or prosthetics for seriously damaged tissue.

[0008] While the methods described above have met with some success, improved methods that accelerate the generation or improve the quality of tissue equivalents would be useful for more rapidly providing usable tissue equivalents. In particular, it would be useful to provide improved methods that promote more rapid acceleration of the cell type of interest and that accelerates the production of extracellular matrix by stromal cells in the tissue equivalent, and also improves the quality of the extracellular matrix produced.

SUMMARY OF THE INVENTION

[0009] The present invention provides methods that increase the production of tissue equivalents that are useful in transplantation therapy, drug testing, cytotoxicity testing of compounds, production of cellular compounds in quantity, and laboratory testing of tissue systems. In one aspect, the present invention provides improved methods for accelerating the production of tissue equivalents by contacting the tissue equivalent with angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments or analogues thereof or AII AT₂ type 2 receptor agonists.

[0010] In another aspect of the present invention, an improved cell culture medium is provided for the production of tissue equivalents, wherein the improvement comprises addition to the culture medium of an effective amount of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments or analogues thereof or AII AT₂ type 2 receptor agonists.

[0011] In a further aspect, the present invention provides kits for the production of tissue equivalents, wherein the kits comprise an effective amount of angiotensinogen, AI, Al analogues, and/or AI fragments and analogues thereof, AII, AII analogues, AII fragments or analogues thereof, and/or AII AT₂ type 2 receptor agonists, and instructions for culturing the tissue equivalents.

BRIEF DESCRIPTION OF THE FIGURES

[0012] FIG. 1 is a graph showing the concentration-dependent effect of AII on epidermal thickness.

[0013] FIG. 2 is a graph showing the effect of exposure to AII during the outgrowth period on the area of an artificial dermis that is covered with keratinocytes.

[0014] FIG. 3 is a graph showing the effect of AII on collagen lattice formation by fibroblasts (750,000 cells per well).

[0015] FIG. 4 is a graph showing the effect of AII on collagen lattice formation by fibroblasts (250,000 cells per well).

[0016] FIG. 5 is a graph showing the effect of on collagen lattice formation by fibroblasts (75,000 cells per well).

[0017] FIG. 6 is a graph showing the effect of AII and AII analogues and fragments on keratinocyte number on integra susbstrate.

[0018] FIG. 7 is a graph showing the effect of AII and AII analogues and fragments on keratinocyte number on Integra susbstrate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] As defined herein, the term “tissue equivalents” refers to three-dimensional cell and tissue culture systems used for the long term proliferation of cells and tissues in vitro in an environment that more closely approximates that found in vivo. Such tissue equivalents include, but are not limited to, equivalents of epithelial tissue, connective tissue, cartilage, bone, organs, vascular grafts, bone marrow, skin, dermis, liver, kidney, cartilage, ligament, tendon, pancreas, heart valve tissues, glands and blood vessels and comprise living cells and extracellular matrix molecules, principally collagen, and may optionally be provided with components not typically found in normal tissue.

[0020] Unless otherwise indicated, the term “active agents” as used herein refers to the group of compounds comprising angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II analogues, AII fragments or analogues thereof and AII AT₂ type 2 receptor agonists.

[0021] U.S. Pat. No. 5,015,629 to DiZerega (the entire disclosure of which is hereby incorporated by reference) describes a method for increasing the rate of healing of wound tissue, comprising the application to such tissue of angiotensin II (AII) in an amount which is sufficient for said increase. The application of AII to wound tissue significantly increases the rate of wound healing, leading to a more rapid re-epithelialization and tissue repair. The term AII refers to an octapeptide present in humans and other species having the sequence Asp-Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:1]. The biological formation of angiotensin is initiated by the action of renin on the plasma substrate angiotensinogen (Circulation Research 60:786-790 (1987); Clouston et al., Genomics 2:240-248 (1988); Kageyama et al., Biochemistry 23:3603-3609; Ohkubo et al., Proc. Natl. Acad. Sci. 80:2196-2200 (1983); all references hereby incorporated in their entirety). The substance so formed is a decapeptide called angiotensin I (AI) which is converted to AII by the converting enzyme angiotensinase which removes the C-terminal His-Leu residues from AI [SEQ ID NO:37]. AII is a known pressor agent and is commercially available.

[0022] Studies have shown that AII increases mitogenesis and chemotaxis in cultured cells that are involved in wound repair, and also increases their release of growth factors and extracellular matrices (dizerega, U.S. Pat. No. 5,015,629; Dzau et. al., J. Mol. Cell. Cardiol. 21:S7 (Supp III) 1989; Berk et. al., Hypertension 13:305-14 (1989); Kawahara, et al., BBRC 150:52-9 (1988); Naftilan, et al., J. Clin. Invest. 83:1419-23 (1989); Taubman et al., J. Biol. Chem. 264:526-530 (1989); Nakahara, et al. BBRC 184:811-8 (1992); Stouffer and Owens, Circ. Res. 70:820 (1992); Wolf, et al., Am. J. Pathol. 140:95-107 (1992); Bell and Madri, Am. J. Pathol. 137:7-12 (1990).

[0023] In addition, AII was shown to be angiogenic in rabbit corneal eye and chick chorioallantoic membrane models (Fernandez, et al., J. Lab. Clin. Med. 105:141 (1985); LeNoble, et al., Eur. J. Pharmacol. 195:305-6 (1991). Additionally, AII and angiotensin III analogs and fragments thereof have been shown to be effective in tissue repair. (U.S. Pat. No. 5,629,292; International Application No. WO 95/08565; International Application WO 95/08337; International Application No. WO 96/39164; all references hereby incorporated in their entirety.)

[0024] Although AII has been shown to increase the proliferation of a number of cell types in vitro, it does not necessarily increase the proliferation of all cell types. AII has been shown to increase cellular proliferation in hair follicles in the area of a thermal injury. (Rodgers et al., J. Burn Care Rehabil. 18:381-388 (1997). The effect of AII on a given cell type has been hypothesized to be dependent, in part, upon the AII receptor subtypes the cell expresses (Shanugam et al., Am. J. Physiol. 268:F922-F930 (1995); Helin et al., Annals of Medicine 29:23-29 (1997); Bedecs et al., Biochem J. 325:449-454 (1997)). These studies have shown that AII receptor subtype expression is a dynamic process that changes during development, at least in some cell types (Id.) While the preceding studies suggest that AII and other AII receptor agonists may accelerate wound repair through increased neovascularization, growth factor release, reepithelialization and/or production of extracellular matrix, the effect of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AI, AII analogues, AII fragments or analogues thereof or AII AT₂ type 2 receptor agonists on accelerating the generation of tissue equivalents is unknown.

[0025] A peptide agonist selective for the AT2 receptor (AII has 100 times higher affinity for AT2 than AT1) has been identified. This peptide is p-aminophenylalanine6-AII [“(p-NH2-Phe)6-AII)”], Asp-Arg-Val-Tyr-Ile-Xaa-Pro-Phe [SEQ ID NO.36] wherein Xaa is p-NH₂-Phe (Speth and Kim, BBRC 169:997-1006 (1990). This peptide gave binding characteristics comparable to AT2 antagonists in the experimental models tested (Catalioto, et al., Eur. j Pharmacol. 256:93-97 (1994); Bryson, et al., Eur. J. Pharmacol. 225:119-127 (1992).

[0026] The effects of AII receptor and AII receptor antagonists have been examined in two experimental models of vascular injury and repair which suggest that both AII receptor subtypes (AT1 and AT2) play a role in wound healing (Janiak et al., Hypertension 20:737-45 (1992); Prescott, et al., Am. J Pathol. 139:1291-1296 (1991); Kauffinan, et al., Life Sci. 49:223-228 (1991); Viswanathan, et al., Peptides 13:783-786 (1992); Kimura, et al., BBRC 187:1083-1090 (1992).

[0027] Many studies have focused upon AII(1-7) (AII residues 1-7) or other fragments of AII to evaluate their activity. AII(1-7) elicits some, but not the full range of effects elicited by AII. Pfeilschifter, et al., Eur. J Pharmacol. 225:57-62 (1992); Jaiswal, et al., Hypertension 19(Supp. II):II-49-II-55 (1992); Edwards and Stack, J. Pharmacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et al., J. Pharmacol. Exper. Ther. 265:664-673 (1991); Jaiswal, et al., Hypertension 17:1115-1120 (1991); Portsi, et a., Br. J. Pharmacol. 111:652-654 (1994).

[0028] As hereinafter defined, a preferred class of AT2 agonists for use in accordance with the present invention comprises AI, AI analogues, AI fragments and analogues thereof, AII, AII analogues or active fragments thereof having p-NH-Phe in a position corresponding to a position 6 of AII. In addition to peptide agents, various nonpeptidic agents (e.g., peptidomimetics) having the requisite AT2 agonist activity are further contemplated for use in accordance with the present invention.

[0029] The active AI, AI analogues, AI fragments and analogues thereof, AII analogues, fragments of AII and analogues thereof of particular interest in accordance with the present invention are characterized as comprising a sequence consisting of at least three contiguous amino acids of groups R¹-R⁸ in the sequence of general formula I

R¹-R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸

[0030] in which R¹ and R² together form a group of formula

X-R^(A)-R^(B)-,

[0031] wherein X is H or a one to three peptide group,

[0032] R^(A) is suitably selected from Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me²Gly, Pro, Bet, Glu(NH₂), Gly, Asp(NH₂) and Suc,

[0033] R^(B) is suitably selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;

[0034] R³ is selected from the group consisting of Val, Ala, Leu, Lys, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;

[0035] R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser, Ala, homoSer and azaTyr;

[0036] R⁵ is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;

[0037] R⁶ is His, Arg or 6-NH₂-Phe;

[0038] R⁷ is Pro or Ala; and

[0039] R⁸ is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R⁴ as a terminal Tyr group.

[0040] Compounds falling within the category of AT2 agonists useful in the practice of the invention include the AII analogues set forth above subject to the restriction that R⁶ is p-NH₂-Phe.

[0041] Particularly preferred combinations for R^(A) and R^(B) are Asp-Arg, Asp-Lys, Glu-Arg and Glu-Lys. Particularly preferred embodiments of this class include the following: AII, AIII or AII(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AII(3-8), also known as des1-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3]; AII(1-7), Asp-Arg-Val-Tyr-Ile-His-Pro {SEQ ID NO:4]; AII(2-7). Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:5]; AII(3-7), Val-Tyr-Ile-His-Pro [SEQ ID NO:6]; AII(5-8), Ile-His-Pro-Phe [SEQ ID NO:7]; AII(1-6), Asp-Arg-Val-Tyr-Ile-His [SEQ ID NO:8]; AII(1-5), Asp-Arg-Val-Tyr-Ile [SEQ ID NO:9]; AII(1-4), Asp-Arg-Val-Tyr [SEQ ID NO:10]; and AII(1-3), Asp-Arg-Val [SEQ ID NO:11]. Other preferred embodiments include: Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID NO:12] and Arg-Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO: 13]. Still another preferred embodiment encompassed within the scope of the invention is a peptide having the sequence Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe [SEQ ID NO:31]. AII(6-8), His-Pro-Phe [SEQ ID NO:14] and AII(4-8), Tyr-Ile-His-Pro-Phe [SEQ ID NO:15] were also tested and found not to be effective.

[0042] In a particularly preferred embodiment, the active compounds of the present invention are selected from those comprising the following general formula:

R1-Arg-R2-R3-R4-His-Pro-R5,

[0043] wherein R1 is selected from the group consisting of H, Gly and Asp;

[0044] R2 is selected from the group consisting of Val, Pro, and Acpc;

[0045] R3 is selected from the group consisting of Tyr and Tyr(PO₃)_(2;)

[0046] R4 is selected from the group consisting of Ala, Val, Ile, Leu, and norLeu; and

[0047] R5 is Phe, Ile, or is absent.

[0048] Another class of compounds of particular interest in accordance with the present invention are those of the general formula II

R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸

[0049] in which R² is selected from the group consisting of H, Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys;

[0050] R³ is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;

[0051] R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser, homoSer and azaTyr;

[0052] R⁵ is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;

[0053] R6 is His, Arg or 6-NH₂-Phe;

[0054] R⁷ is Pro or Ala; and

[0055] R⁸ is selected from the group consisting of Phe, Phe(Br), Ile and Tyr.

[0056] A particularly preferred subclass of the compounds of general formula II has the formula

R²-R³-Tyr-R⁵-His-Pro-Phe [SEQ ID NO: 16]

[0057] wherein R², R³ and R⁵ are as previously defined. Particularly preferred is angiotensin III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]. Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His-Pro-Phe [SEQ ID NO: 17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO:18]. The fragment AII(4-8) was ineffective in repeated tests; this is believed to be due to the exposed tyrosine on the N-terminus.

[0058] In the above formulas, the standard three-letter abbreviations for amino acid residues are employed. In the absence of an indication to the contrary, the L-form of the amino acid is intended. Other residues are abbreviated as follows: TABLE 1 Abbreviation for Amino Acids Me²Gly N,N-dimethylglycyl Bet 1-carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt (betaine) Suc Succinyl Phe(Br) p-bromo-L-phenylalanyl azaTyr aza-α'-homo-L-tyrosyl Acpc 1-aminocyclopentane carboxylic acid Aib 2-aminoisobutyric acid Sar N-methylglycyl (sarcosine)

[0059] It has been suggested that AII and its analogues adopt either a gamma or a beta turn (Regoli, et al., Pharmacological Reviews 26:69 (1974). In general, it is believed that neutral side chains in position R³, R⁵ and R⁷ may be involved in maintaining the appropriate distance between active groups in positions R⁴, R⁶ and R⁸ primarily responsible for binding to receptors and/or intrinsic activity: Hydrophobic side chains in positions R³, R⁵ and R⁸ may also play an important role in the whole conformation of the peptide and/or contribute to the formation of a hypothetical hydrophobic pocket.

[0060] Appropriate side chains on the amino acid in position R² may contribute to affinity of the compounds for target receptors and/or play an important role in the conformation of the peptide. For this reason, Arg and Lys are particularly preferred as R2.

[0061] For purposes of the present invention, it is believed that R³ may be involved in the formation of linear or nonlinear hydrogen bonds with R⁵ (in the gamma turn model) or R⁶ (in the beta turn model). R³ would also participate in the first turn in a beta antiparallel structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it appears that beta and gamma branching are equally effective in this position. Moreover, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R³ may suitably be selected from Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr. Furthermore, Lys has surprisingly been found to be suitable at R³ (see Examples).

[0062] With respect to R⁴, conformational analyses have suggested that the side chain in this position (as well as in R³ and R⁵) contribute to a hydrophobic cluster believed to be essential for occupation and stimulation of receptors. Thus, R⁴ is preferably selected from Tyr, Thr, Tyr (PO₃)₂, homoSer, Ser and azaTyr. Furthermore, Ala has surprisingly been found to be suitable at the R⁴ position (See Examples). In this position, Tyr is particularly preferred as it may form a hydrogen bond with the receptor site capable of accepting a hydrogen from the phenolic hydroxyl (Regoli, et al. (1974), supra).

[0063] In position R⁵, an amino acid with a β aliphatic or alicyclic chain is particularly desirable. Therefore, while Gly is suitable in position R⁵, it is preferred that the amino acid in this position be selected from Ile, Ala, Leu, norLeu, Gly and Val.

[0064] In the AI, AI analogues, AI fragments and analogues thereof, AII analogues, fragments and analogues of fragments of particular interest in accordance with the present invention, R⁶ is His, Arg or 6-NH₂-Phe. The unique properties of the imidazole ring of histidine (e.g., ionization at physiological pH, ability to act as proton donor or acceptor, aromatic character) are believed to contribute to its particular utility as R⁶. For example, conformational models suggest that His may participate in hydrogen bond formation (in the beta model) or in the second turn of the antiparallel structure by influencing the orientation of R⁷. Similarly, it is presently considered that R⁷ should be Pro in order to provide the most desirable orientation of R⁸. In position R⁸, both a hydrophobic ring and an anionic carboxyl terminal appear to be particularly useful in binding of the analogues of interest to receptors; therefore, Tyr and especially Phe are preferred for purposes of the present invention.

[0065] Analogues of particular interest include the following: TABLE 2 Angiotensin II Analogues AII Analogue Sequence Name Amino Acid Sequence Identifier Analogue 1 Asp-Arg-Val-Tyr-Val-His-Pro-Phe SEQ ID NO:19 Analogue 2 Asn-Arg-Val-Tyr-Val-His-Pro-Phe SEQ ID NO:20 Analogue 3 Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe SEQ ID NO:21 Analogue 4 Glu-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO:22 Analogue 5 Asp-Lys-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO:23 Analogue 6 Asp-Arg-Ala-Tyr-Ile-His-Pro-Phe SEQ ID NO:24 Analogue 7 Asp-Arg-Val-Thr-Ile-His-Pro-Phe SEQ ID NO:25 Analogue 8 Asp-Arg-Val-Tyr-Leu-His-Pro-Phe SEQ ID NO:26 Analogue 9 Asp-Arg-Val-Tyr-Ile-Arg-Pro-Phe SEQ ID NO:27 Analogue 10 Asp-Arg-Val-Tyr-Ile-His-Ala-Phe SEQ ID NO:28 Analogue 11 Asp-Arg-Val-Tyr-Ile-His-Pro-Tyr SEQ ID NO:29 Analogue 12 Pro-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO:30 Analogue 13 Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe SEQ ID NO:31 Analogue 14 Asp-Arg-Val-Tyr(PO₃)₂-Ile-His-Pro-Phe SEQ ID NO:32 Analogue 15 Asp-Arg-norLeu-Tyr-Ile-His-Pro-Phe SEQ ID NO:33 Analogue 16 Asp-Arg-Val-Tyr-norLeu-His-Pro-Phe SEQ ID NO:34 Analogue 17 Asp-Arg-Val-homoSer-Tyr-Ile-His-Pro-Phe SEQ ID NO:35

[0066] The polypeptides of the instant invention may be synthesized by methods such as those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984) and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) for solution synthesis. The disclosures of the foregoing treatises are incorporated by reference herein.

[0067] In general, these methods involve the sequential addition of protected amino acids to a growing peptide chain (U.S. Pat. No. 5,693,616, herein incorporated by reference in its entirety). Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage. After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.

[0068] In one aspect of the present invention, a method of accelerating the production of tissue equivalents by exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII analogues, AII fragments or analogues thereof or AII AT₂ type 2 receptor agonists (the “active agents”) is disclosed. Experimental conditions for the production of various tissue equivalents have been reported as follows: liver tissue equivalents (U.S. Pat. No. 5,624,840), bone marrow tissue equivalents (U.S. Pat. No. 5,541,107), ligament tissue equivalents (U.S. Pat. No. 5,521,087), kidney tissue equivalents (U.S. Pat. No. 5,516,680), blood vessel tissue equivalents (U.S. Pat. No. 5,256,418), vascular graft equivalents (U.S. Pat. No. 5,628,781 and 5,387,236) and skin tissue equivalents (5,512,475, 4,835,102, and RE 35,399), all references herein incorporated by reference in their entirety.

[0069] In one embodiment, tissue equivalents are prepared according to standard methods (U.S. Pat. Nos. 5,624,840, 5,541,107, 5,521,087, 5,516,680, 5,256,418, 5,512,475, 4,835,102, and RE 35,399) and incubated in the presence of, preferably, between about 0.1 ng/ml and about 1 mg/ml of the active agents of the invention.

[0070] In another embodiment, a dermal equivalent is formed by the inoculation of fibroblasts onto a three-dimensional matrix and their growth to subconfluence (U.S. Pat. No. 5,578,485, incorporated by reference herein in its entirety). The three-dimensional support may be of any material and/or shape that: (a) allows cells to attach to it (or can be modified to allow cells to attach to it); and (b) allows cells to grow in more than one layer. A number of different materials may be used to form the matrix, including but not limited to: nylon (polyamides), dacron (polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE; teflon), thermanox (TPX), nitrocellulose, cotton, polyglycolic acid (PGA), cat gut sutures, cellulose, gelatin, dextran, etc. Any of these materials may be woven into a mesh, for example, to form the three-dimensional matrix. Certain materials, such as nylon, polystyrene, etc., are poor substrates for cellular attachment. When these materials are used as the three-dimensional support matrix, it is advisable to pre-treat the matrix prior to inoculation of stromal cells in order to enhance the attachment of stromal cells to the matrix. For example, prior to inoculation with stromal cells, nylon matrices could be treated with 0.1M acetic acid, and incubated in polylysine, FBS, and/or collagen to coat the nylon. Polystyrene could be similarly treated using sulfuric acid.

[0071] Where the three-dimensional culture is itself to be implanted in vivo, it may be preferable to use biodegradable matrices such as poly glycolic acid, catgut suture material, or gelatin, for example (U.S. Pat. No. 5,578,485). Where the cultures are to be maintained for long periods of time or cryopreserved, non-degradable materials such as nylon, dacron, polystyrene, polyacrylates, polyvinyls, teflons, cotton, etc. may be preferred. A convenient nylon mesh which could be used in accordance with the invention is Nitex, a nylon filtration mesh having an average pore size of 210 mu m and an average nylon fiber diameter of 90 mu m (#3-210/36, Tetko, Inc., N.Y.).

[0072] In a preferred embodiment, the fibroblasts are allowed to proliferate until the entire growth substrate is covered, although only approximately 60% confluency of the fibroblasts on the three-dimensional matrix is required to support the growth of epidermal cells later inoculated. The fibroblasts will continue to divide even after they have reached confluency because the three-dimensional culture permits the exit of cells, thereby preventing contact inhibition. Although any fibroblasts may be utilized in the inoculum, it is advantageous to use skin fibroblasts, as these will deposit the appropriate types of collagen and elaborate other dermal components. Fibroblasts may be allogeneic or autologous. Skin fibroblasts may be readily obtained from cellular suspensions prepared by mechanical and/or enzymatic disaggregation of dermal tissue. When the cellular suspension obtained is plated, the fibroblasts will adhere more quickly than other cells, and thus, can be grown to confluence, lifted by mild enzymatic treatment and inoculated onto the three-dimensional matrix.

[0073] While the use of fibroblasts alone is sufficient to form a three-dimensional stromal matrix that functions as a dermal equivalent, additional types of stromal cells may be used to inoculate the three-dimensional matrix. These include, but are not limited to endothelial cells, pericytes, macrophages, monocytes, lymphocytes, plasma cells, adipocytes, etc.

[0074] In a further preferred embodiment, epidermal cells are inoculated onto the dermal equivalent to provide full thickness skin equivalents (U.S. Pat. No. 5,578,485, incorporated by reference herein in its entirety). To this end, melanocytes and keratinocytes may be inoculated simultaneously, or preferably, in sequence. For example, keratinocytes can be inoculated onto subconfluent melanocytes which were previously inoculated onto the stromal matrix.

[0075] Melanocytes and keratinocytes may be allogeneic or autologous in their relationship to fibroblast stromal cells, can be isolated from skin using known procedures which involve incubating skin in a digestive enzyme, such as trypsin, in order to separate dermal and epidermal layers.

[0076] In one embodiment, keratinocytes and melanocytes may be isolated as follows. A tissue sample, e.g. foreskin, may be trimmed so that the entire surface may be easily exposed to antibiotics. Tissue may be first washed in a concentrated antibiotic solution for twenty minutes, followed by two subsequent washes of ten minutes each. The outer portion of the tissue may then be cut into small pieces, and then placed in a 0.15% trypsin solution (in PBS without calcium or magnesium), quickly removed, placed in a fresh container of the same trypsin solution (such that all the tissue is covered by solution), and refrigerated overnight at between about 2° C. and 8° C. The next day, the tissue pieces may be removed from the trypsin solution, and the epidermis separated from the dermis using curved forceps. The epidermis may be placed in a conical tube, and about 0.15% trypsin in PBS (without calcium or magnesium) may be used to digest the tissue into a single cell suspension; to facilitate this process, the sample may be repeatedly aspirated into and out of a Pasteur pipette. When the sample appears to be a single cell suspension, it may be centrifuged at 1400 g for about 7 minutes and then resuspended in either growth media or in growth media containing 0.01 mg/ml PMA, which selects for melanocytes. Accordingly, cultures of keratinocytes or melanocytes may be produced. The epidermal cells can be suspended and used to inoculate the dermal equivalent. Alternatively, the epidermal cell suspension can be plated and melanocytes and keratinocytes separated based upon their differential attachment qualities. Isolated melanocytes may first be inoculated onto the dermal equivalent and allowed to grow for a few days prior to inoculation of keratinocytes. This “tissue” grows rapidly and can be maintained in nutrient media without exogenous growth factors.

[0077] In a preferred embodiment, a dermal equivalent produced as set forth supra may be inoculated with keratinocytes as follows (U.S. Pat. No. 5,478,739, incorporated by reference herein in its entirety). Fresh dermal equivalent cultures, or dermal equivalent cultures removed from the freezer and rinsed with PBS in order to remove dimethyl sulfoxide (DMSO), may be allowed to equilibrate in stratification medium (DMEM with 5 percent fetal bovine serum; 100 mu g/ml ascorbate (Sigma) and 0.5 mu g/ml hydrocortisone (Sigma)) for about 24-48 hours. Keratinocytes may then be seeded onto the dermal equivalent at a density of about 5×10⁵ keratinocytes per cm of dermal equivalent. The keratinocyte/dermal equivalent co-cultures may then be incubated submerged in stratification medium for 5-7 days, then raised such that keratinocytes may differentiate at the air/liquid interface. After about 12-14 days in culture, a cholesterol-rich lipid supplement (Sigma) (0.5%) may be added to the stratification medium and the cultures may be grown for an additional 12-21 days until a multi-layered stratum corneum is formed.

[0078] In another embodiment, bone marrow cells are grown on a three-dimensional support in co-cultures with stromal cells comprising fibroblasts (of either fetal or bone marrow origin) or a mixture of cell types which comprise the stromal components of normal marrow, including fibroblasts, macrophages, reticular cells, and adipocytes (U.S. Pat. No. 5,541,107). Factors derived from media of splenic and/or hepatic (liver) macrophage cultures or from subsets of stromal cells may optionally be added to the culture. The three-dimensional culture system of the present invention appears to maximize the proliferation of multipotential hematopoietic stem cells which have the capability of repopulating bone marrow when the bone marrow has been destroyed by intrinsically or environmentally-mediated disease or by the treatment of such disease with chemotherapy and/or radiation.

[0079] Alternatively, a liver tissue equivalent is produced by inoculation and culturing of liver parenchymal cells on a pre-established three-dimensional stromal tissue (U.S. Pat. No. 5,624,840). The stromal tissue comprises stromal cells grown on a three-dimensional matrix or framework. The stromal cells comprise fibroblasts with or without additional cells and/or elements. The fibroblasts and other cells and/or elements that comprise the stroma may be fetal or adult in origin, and may be derived from convenient sources such as skin, liver, pancreas, etc. Such tissues and/or organs can be obtained by appropriate biopsy or upon autopsy. In fact, cadaver organs may be used to provide a generous supply of stromal cells and elements.

[0080] In another embodiment, bone, ligament, cartilage and tendon equivalents, are produced by forming a collagen gel having living connective tissue cells dispersed therein (U.S. Pat. No. 5,521,087). The cells are capable of contracting the gel. The gel is maintained under conditions suitable for contraction by the connective tissue cells, while simultaneous contraction of the gel is restrained to define an axis of predetermined length for cell alignment. The connective tissue cells align along the defined axis to produce an oriented tissue-equivalent having increased mechanical strength in the direction of the axis.

[0081] In another embodiment, kidney tissue equivalents are prepared by culturing kidney parenchymal cells cultured on a living stromal tissue prepared in vitro, said living stromal tissue comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells (U.S. Pat. No. 5,516,680).

[0082] In another embodiment, pancreatic tissue equivalents are formed by mincing pancreatic tissue and washing in calcium-free, magnesium-free buffer (U.S. Pat. No. 5,578,485, incorporated by reference herein in its entirety.) The minced tissue fragments are incubated in a solution of trypsin and collagenase. Dissociated cells may be filtered using a 20 mu m nylon mesh, resuspended in a suitable buffer such as Hanks balanced salt solution, and pelleted by centrifugation. The resulting pellet of cells can be resuspended in minimal amounts of appropriate media and inoculated onto the three-dimensional stroma.

[0083] In another embodiment, living vascular graft equivalents, comprising a polymeric backbone and endothelial cells are prepared as described in U.S. Pat. Nos. 5,628,781 and 5,387,236, both references hereby incorporated in their entirety. In a preferred embodiment, a polymeric substrate for the vascular prosthesis is dispersed in a solution of biological tissue fragments such as vascular tissues, connective tissues, fat tissues and muscular tissues and/or cells such as vascular endothelial cells, smooth muscle cells and fibroblast cells. (U.S. Pat. No. 5,387,236) The cells and/or tissue fragments are deposited and captured within the wall and on the inner surface of the vascular prosthesis substrate wall from the outside and/or the inside of the vascular prosthesis substrate by providing a pressure differential between the outside and the inside of the substrate. The interior space of the vascular prosthesis can be vacuumized or pressurized, and the cells and/or tissue fragments are deposited and captured in and on the walls either outside or inside of the prosthesis substrate. In a preferred embodiment, the living vascular graft further comprises vascular smooth muscle cells and fibroblasts.

[0084] Methods for producing heart valve equivalents are described in U.S. Pat. Nos. 5,713,950; 5,480,424; 5,192,312; and 5,052,934, all references herein incorporated by reference in their entirety.

[0085] In order to accelerate the generation of the all of the above tissue equivalents, they are exposed to preferably, between about 0.1 ng/ml and about 1 mg/ml of the active agents of the invention as described above. Acceleration of tissue equivalent generation by exposure to the lo active agents occurs via increased proliferation of the cells that comprise the tissue equivalent, increase in growth factor production by cells of the tissue equivalent, and via increasing production of extracellular matrix components by stromal cells within the tissue equivalents.

[0086] Proliferation of cells in the tissue equivalent can be quantitated using any one of a variety of techniques well known in the art, including, but not limited to, bromodeoxyuridine incorporation (Vicario-Abejon et al., 1995), ³H-thymidine incorporation (Fredericksen et al., 1988), or antibody labeling of a protein present in higher concentration in proliferating cells than in non-proliferating cells. In a preferred embodiment, accelerated production of tissue equivalents is assessed by antibody detection of a protein known to be present in higher concentrations in proliferating cells than in non-proliferating cells, including but not limited to proliferating cell nuclear antigen (PCNA, or cyclin; Zymed Laboratories, South San Francisco, Calif.).

[0087] Increased production of growth factors, including but not limited to transforming growth factor beta, fibroblast growth factor, and epidermal growth factor, by cells of the tissue equivalent can be quantitated by standard immunohistochemical techniques using antibodies to the growth factors (DAKO, Carpernterica, Calif.; Genzyme, Cambridge, Mass.; Sigma Chemical Co., St. Louis, Mo.).

[0088] Increasing production of extracellular matrix components by the stromal component of the tissue equivalent can be quantitated by antibody labeling of extracellular matrix components, including but not limited to fibronectin, elastin, glycosaminoglycans, and laminin (DAKO, Carpernterica, Calif.; Pharmingen, San Diego, Calif.).

[0089] In another aspect of the present invention, an improved cell culture medium is provided for accelerated production of tissue equivalents, wherein the improvement comprises addition to the cell culture medium of an effective amount of active agents, as described above. For any given active agent, the optimum concentration for a given formulation may be readily determined empirically. In general, a concentration of active agent suitable for use in accordance with the present invention preferably ranges between about 0.1 ng/ml and about 1 mg/ml active agents.

[0090] Any cell culture media that can support the growth of tissue equivalents can be used with the present invention. Such cell culture media include, but are not limited to Basal Media Eagle, Dulbecco's Modified Eagle Medium, Iscove's Modified Dulbecco's Medium, McCoy's Medium, Minimum Essential Medium, F-10 Nutrient Mixtures, Opti-MEM® Reduced-Serum Medium, RPMI Medium, and Macrophage-SFM Medium or combinations thereof. In a preferred embodiment, the defined cell culture medium described in U.S. Pat. No. 5,712,163 is used.

[0091] The improved cell culture medium can be supplied in either a concentrated (ie: 10×) or non-concentrated form, and may be supplied as either a liquid, a powder, or a lyophilizate. The cell culture may be either chemically defined, or may contain a serum supplement. Culture media is commercially available from many sources, such as GIBCO BRL (Gaithersburg, Md.) and Sigma (St. Louis, Mo.).

[0092] In a further aspect, the present invention provides kits for the propagation of tissue equivalents, wherein the kits comprise an effective amount of the active agents of the invention, and instructions for their use in accelerating the production of tissue equivalents.

[0093] In a preferred embodiment, the kit further comprises cell culture growth medium. Any cell culture media that can support the growth of tissue equivalents can be used with the present invention. Examples of such cell culture media are described above.

[0094] The improved cell culture medium can be supplied in either a concentrated (ie: 10×) or non-concentrated form, and may be supplied as a liquid, a powder, or a lyophilizate. The cell culture may be either chemically defined, or may contain a serum supplement. In a further embodiment, the kit further comprises a three-dimensional support material.

EXAMPLE 1 Effect of AII on Epidermal Layer Thickness

[0095] The purpose of this study was to determine, by quantitative histology, the effect of presoaking a living skin equivalent (LSE) in a lactated Ringer's solution (LRS)-Dextrose containing AII, on the thickness of the epidermis on day 23 post-grafting.

[0096] Twenty three male Swiss nude mice (22-24 grams) were purchased from Taconic Laboratories and quarantined at least two days prior to surgery, and divided into 4 groups of mice. The mice were anesthetized with an intramuscular injection of Ketamine (ketaset; Phoenix Pharmaceuticals, Inc.) and Rompun (xylazine; Phoenix Pharmaceuticals, Inc.) and a 1 cm×1 cm full thickness skin excision was made on their dorsal surface. Each group received an Apligraph LSE (Organogenesis; Canton, Mass.) that had been soaked for 10 minutes in concentrations of AII ranging from 0 to 1.0 mg/ml. The LSE was placed in the defect and trimmed with microscissors so that no gap was observed between the edges of the mouse skin and the LSE. After the graft was placed, the dorsal surface of the mouse was covered by petroleum embedded gauze (Dermacea) followed by two adhesive bandages (Baxter). After recovery from anesthesia, the mice were returned to their individual cages and observed daily until euthenasia. The mice received intramuscular analgesia for the first three days after surgery. No mouse lost their bandage prior to bandage removal on day 7 and day 23. At necropsy, the degree of graft taken and the appearance of the grafted tissue was noted prior to placement of the biopsy in 10% buffered formalin in preparation for paraffin embedding and sectioning for hematoxylin and eosin staining.

[0097] AII grafts appeared healthy (except one control which had lost its graft) and inosculation was noted for 80-100% of the graft edges. On day 7, one of the AII-treated grafts had numerous vessels attached to its underside (against the fascia of the nude mice after full thickness excision). This was not noted on any of the other mice.

[0098] Measurement of the thickness of the epidermis at day 23 was accomplished with an ocular micrometer in a 10× ocular at the 10× magnification on the objective (100× total magnification). The thickest part of each graft was measured and the thickness of the graft at one-half of a 10× field fo each side of the thickest portion was also measured. As can be seen in FIG. 1, the epidermal thickness increased in a concentration-dependent manner after AII administration.

Example 2 Effect of AII on the Outgrowth of Keratinocytes from Explants of Human Skin

[0099] Human skin explants were obtained from surgery of split thickness skin grafts and cut by scalpel in to 2 mm×2 mm squares. Ten explants per condition were placed dermal side down on frozen and thawed Dermagraft (Advanced Tissue Sciences, San Diego, Calif.) and cultured at the air-liquid interface using Keratinocyte-SFM medium (Gibco BRL, Grand Island, N.Y.) supplemented with epidermal growth factor and bovine pituitary extract according to the manufacturer's instructions. The culture medium contained penicillin (50 U/ml) and streptomycin (50 μg/ml) and extra calcium chloride to a final concentration of 1 mM. Angiotensin II was added to the culture medium at a final concentration of 1 or 10 μg/ml. The cultures were maintained at 37° C. with 5% CO₂ and medium with and without AII and the medium was changed twice weekly. After 7 days, the cultures were fixed in formalin for one hour, stained with hematoxylin and washed in tap water. The dermal replacements appeared dark purple except for the area where the keratinocytes exclude the stain. Cultures were photographed using a dissection microscope at a fixed magnification and the area of outgrowth was quantified by gravimetric planimetry. As shown in FIG. 2, exposure to AII during the outgrowth period increased the area of the artificial dermis covered with keratinocytes.

EXAMPLE 3 Contraction of Collagen Lattices by Human Fibroblasts

[0100] Contraction of collagen lattices is a first step in the preparation of an artificial dermis, where keratinocytes are subsequently grown in the collagen matrix. Collagen lattices can also be implanted sub-cutaneously and used as a bulking-up agent for plastic surgery applications.

[0101] Normal human fibroblasts were purchased from Clonetics (San Diego, Calif.) and were thawed and cultured in Fibroblast Growth Medium (Clonetics) according to the manufacturer's instructions. Once the cells reached confluence in the flask, they were harvested by trypsinization and utilized for the studies described below.

[0102] Rat tails were harvested from 200 gram female rats and frozen until use. The frozen rat tails were thawed in 70% (vol/vol) ethanol for 20 minutes. The tendon bundles were excised in 70% ethanol in a vertical laminar flow hood. The individual tendons were pulled out of the tendon sheath, minced, and placed in dilute acetic acid (1:1000) using 250 ml per tail. The mixture was incubated at 4° C. for 48 hours, at which point the minced tendons had swelled to the volume of the dilute acetic acid. The viscous mixture was centrifuged at 23,000 rpm in a Beckman L ultracentrifuge for 1 hour. The supernatant was harvested and further refined. This crude collagen solution was mixed with 0.1M NaOH in a 6:1 ratio to neutralize the acetic acid and precipitate the collagen. The mixture was then centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and an equal volume of fresh acetic acid (1:1000) was introduced to resolubilize the collagen over 48 hours. This solution was stored at 4° C. as refined collagen. The protein concentration was determined by BCA assay.

[0103] The collagen lattices for the assessment of contraction by fibroblasts and angiotensin II were formed in 60 mm Falcon bacteriological dishes. Each dish contained 1 ml 4× DMEM High glucose medium, 1 ml fetal calf serum, 0.25 ml NaOH, 1.5 ml of 500 μg/ml refined collagen and 1 ml of fibroblasts in Fibroblast Growth Medium (7.5×10⁴ cells/ml to 7.5×10⁵ cells/ml). In these cultures, various concentrations of AII (1 to 10 μg/ml) were added to assess the effect of AII on the formation of an artificial dermis. The cultures were placed in an incubator at 37° C. in an atmosphere of 5% CO₂ in air. At various times after culture initiation, the diameter of the formed lattice was measured. As there are slight differences in diameter at various points (ie: the lattices were not always perfectly round), the average of the largest and the smallest diameters were taken. As shown in FIGS. 3-5, AII accelerated the contraction of collagen lattices.

[0104] Similar experiments were conducted using AII analogues and fragments, except that ten μg/ml of each of the peptides shown in Table 3 were added to 1×10⁵ of fibroblasts/well. At various times after culture initiation, the diameter of the formed lattice was measured. The results of these experiments are shown in Table 4, and demonstrate that each of the AII analogues and fragments accelerated the contraction of collagen lattices. TABLE 3 Designation for Analogues/Fragments Abbreviation Sequence SEQ ID NO: Gly¹-AII GRVYIHPF SEQ ID NO:39 NorLeu⁴-AIII --RVYnLHPF SEQ ID NO:40 Acpc³-AII DR(Acpc)YIHPF SEQ ID NO:4l Ile AII DRVYIHPI SEQ ID NO:38 Ala⁴-AIII --RVYAHPF SEQ ID NO:18 AII(1-7) DRVYIHP-- SEQ ID NO:4 AII DRVYIHPF SEQ ID NO.1

[0105] TABLE 4 Results of Collagen Lattice Contraction with AII Analogues and Fragments Day of Culture Diameter of Lattice (cm) Peptide Day 1 Day 2 Day 3 Day 4 None 3.5 2.8 2.3 1.9 Ala4-AIII 3.4 2.7 1.9 1.6 Gly1 AII 3.4 2.2 1.6 1.5 NorLeu4 AIII 3.4 2.0 1.6 1.5 Acpc3 AII 3.5 2.0 1.6 1.4 AII(1-7) 3.4 2.4 2.0 1.7 AII 3.3 2.4 1.6 1.4 Ile8 AII 3.4 2.4 1.7 1.4

EXAMPLE 4 Effect of AII, AII Analogues and Fragments, and AII Fragment Analogues on Keratinocyte Number on an Artificial Dermis

[0106] Integra, a commercially available artificial dermis of chondrotin sulfate and collagen, was obtained (Integra Life Sciences) and used in culture as a matrix for keratinocyte growth. The Integras was washed free of preservative by a sterile saline for injection and cut to size to fit snugly into the bottom of 24 well plates. After trimming, the pieces of Integra were placed silicone-side down into the wells.

[0107] Human keratinocytes were purchased from Clonetics and thawed and cultured as described in Example 3, except that they were grown in Keratinocyte Growth Medium, as per the manufacturer's instructions. Once the cells reached confluence in the flask, they were detached from the tissue culture flasks by trypsinization. The cells were resuspended at 1×10⁵ cells/ml in Keratinocyte Growth Medium or Keratinocyte Basal Medium with or without 10 μg/ml of various AII-related peptides (AII, AII(1-7), Pro3 AII(1-7), Ala4-AIII, and Pro3-AII; see Table 5 below). One ml of these cell preparations was added to wells of the 24 well plates containing Integra membrane. The cultures were placed in an incubator at 37° C. in an atmosphere of 5% CO₂ in air. At various times after initiation of the cultures, the number of keratinocytes on the surface of the Integra membrane in 1 500× fields was assessed under phase contrast microscopy. The data are summarized in FIGS. 6-7 and demonstrate that each of the peptides tested increased the proliferation of keratinocytes on this artificial membrane. TABLE 5 Designation for Analogues/Fragments Name Abbreviation Sequence SEQ ID NO: GSD 24B Pro³-AII DRPYIHPF SEQ ID NO:31 2GD Pro³-AII(1-7) DRPYIHP SEQ ID NO:42 GSD 22A Ala⁴-AIII  RVYAHIPF SEQ ID NO:18 AII(1-7) DRVYIHP-- SEQ ID NO:4 AII DRVYIHPF SEQ ID NO.1

[0108] The present invention, by providing a method for enhanced production of tissue equivalents, will greatly increase the clinical benefits of tissue equivalent transplantation, as well as increasing the utility of drug and cytotoxicity testing on tissue equivalents, production of cellular compounds in quantity, and laboratory testing of tissue equivalent systems.

[0109] The present invention is not limited by the aforementioned particular preferred embodiments. It will occur to those ordinarily skilled in the art that various modifications may be made to the disclosed preferred embodiments without diverting from the concept of the invention. All such modifications are intended to be within the scope of the present invention.

1 42 1 8 PRT Artificial Sequence Description of Artificial SequenceAII 1 Asp Arg Val Tyr Ile His Pro Phe 1 5 2 7 PRT Artificial Sequence Description of Artificial SequenceAII (2-8) 2 Arg Val Tyr Ile His Pro Phe 1 5 3 6 PRT Artificial Sequence Description of Artificial SequenceAII (3-8) 3 Val Tyr Ile His Pro Phe 1 5 4 7 PRT Artificial Sequence Description of Artificial SequenceAII (1-7) 4 Asp Arg Val Tyr Ile His Pro 1 5 5 6 PRT Artificial Sequence Description of Artificial SequenceAII (2-7) 5 Arg Val Tyr Ile His Pro 1 5 6 5 PRT Artificial Sequence Description of Artificial SequenceAII (3-7) 6 Val Tyr Ile His Pro 1 5 7 4 PRT Artificial Sequence Description of Artificial SequenceAII (5-8) 7 Ile His Pro Phe 1 8 6 PRT Artificial Sequence Description of Artificial SequenceAII (1-6) 8 Asp Arg Val Tyr Ile His 1 5 9 5 PRT Artificial Sequence Description of Artificial SequenceAII (1-5) 9 Asp Arg Val Tyr Ile 1 5 10 4 PRT Artificial Sequence Description of Artificial SequenceAII (1-4) 10 Asp Arg Val Tyr 1 11 3 PRT Artificial Sequence Description of Artificial SequenceAII (1-3) 11 Asp Arg Val 1 12 7 PRT Artificial Sequence Description of Artificial SequenceAII analogue 12 Arg Xaa Tyr Ile His Pro Phe 1 5 13 7 PRT Artificial Sequence Description of Artificial SequenceAII analogue 13 Arg Val Tyr Xaa His Pro Phe 1 5 14 3 PRT Artificial Sequence Description of Artificial SequenceAII (6-8) 14 His Pro Phe 1 15 5 PRT Artificial Sequence Description of Artificial SequenceAII (4-8) 15 Tyr Ile His Pro Phe 1 5 16 7 PRT Artificial Sequence Description of Artificial SequenceAII analogue class 16 Xaa Xaa Tyr Xaa His Pro Phe 1 5 17 7 PRT Artificial Sequence Description of Artificial SequenceAII analogue 17 Arg Val Tyr Gly His Pro Phe 1 5 18 7 PRT Artificial Sequence Description of Artificial SequenceAII analogue 18 Arg Val Tyr Ala His Pro Phe 1 5 19 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 1 19 Asp Arg Val Tyr Val His Pro Phe 1 5 20 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 2 20 Asn Arg Val Tyr Val His Pro Phe 1 5 21 11 PRT Artificial Sequence Description of Artificial SequenceAII analogue 3 21 Ala Pro Gly Asp Arg Ile Tyr Val His Pro Phe 1 5 10 22 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 4 22 Glu Arg Val Tyr Ile His Pro Phe 1 5 23 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 5 23 Asp Lys Val Tyr Ile His Pro Phe 1 5 24 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 6 24 Asp Arg Ala Tyr Ile His Pro Phe 1 5 25 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 7 25 Asp Arg Val Thr Ile His Pro Phe 1 5 26 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 8 26 Asp Arg Val Tyr Leu His Pro Phe 1 5 27 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 9 27 Asp Arg Val Tyr Ile Arg Pro Phe 1 5 28 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 10 28 Asp Arg Val Tyr Ile His Ala Phe 1 5 29 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 11 29 Asp Arg Val Tyr Ile His Pro Tyr 1 5 30 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 12 30 Pro Arg Val Tyr Ile His Pro Phe 1 5 31 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 13 31 Asp Arg Pro Tyr Ile His Pro Phe 1 5 32 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 14 32 Asp Arg Val Tyr Ile His Pro Phe 1 5 33 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 15 33 Asp Arg Xaa Tyr Ile His Pro Phe 1 5 34 8 PRT Artificial Sequence Description of Artificial SequenceAII analogue 16 34 Asp Arg Val Tyr Xaa His Pro Phe 1 5 35 9 PRT Artificial Sequence Description of Artificial SequenceAII analogue 17 35 Asp Arg Val Ser Tyr Ile His Pro Phe 1 5 36 8 PRT Artificial Sequence Description of Artificial Sequencep-aminophenylalanine 6 AII 36 Asp Arg Val Tyr Ile Xaa Pro Phe 1 5 37 10 PRT Artificial Sequence Description of Artificial Sequenceangiotensin I 37 Asp Arg Val Tyr Ile His Pro Phe His Leu 1 5 10 38 8 PRT Artificial Sequence Description of Artificial Sequence1GDIle8-AII 38 Asp Arg Val Tyr Ile His Pro Ile 1 5 39 8 PRT Artificial Sequence Description of Artificial SequenceGly1-AII 39 Gly Arg Val Tyr Ile His Pro Phe 1 5 40 7 PRT Artificial Sequence Description of Artificial SequencenorLeu4-AIII 40 Arg Val Tyr Xaa His Pro Phe 1 5 41 8 PRT Artificial Sequence MOD_RES (3) Xaa at position 3 is 1-aminocyclopentane carboxylic acid 41 Asp Arg Xaa Tyr Ile His Pro Phe 1 5 42 7 PRT Artificial Sequence Description of Artificial Sequence2GD Pro3-AII(1-7) 42 Asp Arg Pro Tyr Ile His Pro 1 5 

We claim:
 1. An improved method for producing a tissue equivalent, the improvement comprising contacting the tissue equivalent with an amount effective to accelerate generation of tissue equivalents of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R¹-R⁸ in the sequence of general formula I R¹-R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸ in which R¹ and R² together form a group of formula X-R^(A)-R^(B)-, wherein X is H or a one to three peptide group R^(A) is selected from Asp, Glu, Asn, Acpc, Ala, Me²Gly, Pro, Bet, Glu(NH₂), Gly, Asp(NH₂) and Sue; R^(B) is selected from Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys; R³ is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys and Tyr; R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser, Ala, homoSer and azaTyr; R⁵ is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly; R⁶ is His, Arg or 6-NH₂-Phe; R⁷ is Pro or Ala; and R⁸ is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R⁴ as a terminal Tyr group.
 2. The method of claim 1 wherein the active agent is selected from the group consisting of angiotensinogen, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42.
 3. The method of claim 1 wherein the active agent is SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:31 SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.
 4. The method of claim 1 wherein the concentration of active agent is between about 0.1 ng/kg and about 1.0 mg/kg.
 5. The method of claim 1 where the tissue equivalent is selected from the group consisting of a skin, dermis, bone, bone marrow, pancreas, heart valve, vascular graft, cartilage, ligament, liver, and kidney tissue equivalent.
 6. An improved chemically defined medium for the culture of tissue equivalents, wherein the improvement comprises contacting the tissue equivalent with an amount effective to accelerate generation of tissue equivalents of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R¹-R⁸ in the sequence of general formula I R¹-R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸ in which R¹ and R² together form a group of formula X-R^(A)-R^(B)-, wherein X is H or a one to three peptide group R^(A) is selected from Asp, Glu, Asn, Acpc, Ala, Me²Gly, Pro, Bet, Glu(NH₂), Gly, Asp(NH2) and Suc; R^(B) is selected from Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys; R³ is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys and Tyr; R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser, Ala, homoSer and azaTyr; R⁵ is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly; R⁶ is His, Arg or 6-NH₂-Phe; R⁷ is Pro or Ala; and R⁸ is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R⁴ as a terminal Tyr group.
 7. The improved chemically defined medium of claim 6 wherein the active agent is selected from the group consisting of angiotensinogen, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42.
 8. The improved chemically defined medium of claim 6 wherein the active agent is SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:31,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.
 9. The improved chemically defined medium of claim 6 wherein the concentration of active agent is between about 0.1 ng/ml and about 1.0 mg/ml.
 10. The improved chemically defined medium of claim 6 where the tissue equivalent is selected from the group consisting of a skin, dermis, bone, bone marrow, pancreas, heart valve, vascular graft, cartilage, ligament, liver, and kidney tissue equivalent.
 11. An improved kit for the culture of tissue equivalents, wherein the improvement comprises providing a). an amount effective to accelerate generation of tissue equivalents of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R¹-R⁸ in the sequence of general formula I R¹-R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸ in which R¹ and R² together form a group of formula X-R^(A)-R^(B)-, wherein X is H or a one to three peptide group R^(A) is selected from Asp, Glu, Asn, Acpc, Ala, Me²Gly, Pro, Bet, Glu(NH₂), Gly, Asp(NH₂) and Suc; R^(B) is selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys; R³ is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys and Tyr; R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser, Ala, homoSer and azaTyr; R⁵ is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly; R⁶ is His, Arg or 6-NH₂-Phe; R⁷ is Pro or Ala; and R⁸ is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R⁴ as a terminal Tyr group; and b) and instructions for use of the active agent to accelerate generation of tissue equivalents.
 12. The kit of claim 11 further comprising tissue culture medium.
 13. The kit claim 11 wherein the active agent is selected from the group consisting of angiotensinogen, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42.
 14. The kit of claim 11 wherein the active agent is SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:31, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.
 15. The kit of claim 11 wherein the concentration of active agent is between about 0.1 ng/ml and about 1.0 mg/ml.
 16. An improved method for producing a tissue equivalent, the improvement comprising contacting the tissue equivalent with an amount effective to accelerate generation of tissue equivalents of at least one active agent comprising a sequence of the following general formula: R1-Arg-R2-R3-R4-His-Pro-R5 wherein R1 is selected from the group consisting of H, Gly and Asp; R2 is selected from the group consisting of Val, Pro, and Acpc; R3 is selected from the group consisting of Tyr and Tyr(PO₃)₂; R4 is selected from the group consisting of Ala, Val, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent.
 17. The method of claim 16 wherein the active agent is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.
 18. The method of claim 16 wherein the concentration of active agent is between about 0.1 ng/kg and about 1.0 mg/kg.
 19. The method of claim 16 where the tissue equivalent is selected from the group consisting of a skin, dermis, bone, bone marrow, pancreas, heart valve, vascular graft, cartilage, ligament, collagen lattice, liver, and kidney tissue equivalent.
 20. The method of claim 16 where the tissue equivalent is selected from the group consisting of a collagen lattice and dermis tissue equivalent.
 21. An improved chemically defined medium for the culture of tissue equivalents, wherein the improvement comprises contacting the tissue equivalent with an amount effective to accelerate generation of tissue equivalents of at least one active agent comprising a sequence of the following general formula: R1-Arg-R2-R3-R4-His-Pro-R5 wherein R is selected from the group consisting of H, Gly and A sp; R2 is selected from the group consisting of Val, Pro, and Acpc; R3 is selected from the group consisting of Tyr and Tyr(PO₃)₂; R4 is selected from the group consisting of Ala, Val, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent.
 22. The chemically defined medium of claim 21 wherein the active agent is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.
 23. The method of claim 21 wherein the concentration of active agent is between about 0.1 ng/kg and about 1.0 mg/kg.
 24. The method of claim 21 where the tissue equivalent is selected from the group consisting of a skin, dermis, bone, bone marrow, pancreas, heart valve, vascular graft, cartilage, ligament, collagen lattice, liver, and kidney tissue equivalent.
 25. The method of claim 21 where the tissue equivalent is selected from the group consisting of a collagen lattice and dermis tissue equivalent.
 26. An improved kit for the culture of tissue equivalents, wherein the improvement comprises providing a). an amount effective to accelerate generation of tissue equivalents of at least one active agent comprising a sequence of the following general formula: R1-Arg-R2-R3-R4-His-Pro-R5 wherein R1 is selected from the group consisting of H, Gly and Asp; R2 is selected from the group consisting of Val, Pro, and Acpc; R3 is selected from the group consisting of Tyr and Tyr(PO₃)₂; R4 is selected from the group consisting of Ala, Val, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent; and b) instructions for use of the active agent to accelerate generation of tissue equivalents.
 27. The kit of claim 26 wherein the active agent is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.
 28. The kit of claim 26 wherein the concentration of active agent is between about 0.1 ng/kg and about 1.0 mg/kg.
 29. The kit of claim 26 where the tissue equivalent is selected from the group consisting of a skin, dermis, bone, bone marrow, pancreas, heart valve, vascular graft, cartilage, ligament, collagen lattice, liver, and kidney tissue equivalent.
 30. The kit of claim 16 where the tissue equivalent is selected from the group consisting of a collagen lattice and dermis tissue equivalent. 